The present invention relates to an automatic electrophysiological measuring apparatus and method using Xenopus Oocytes.
The science of inquiring into the electrical properties of living organisms is known as xe2x80x9celectrophysiology,xe2x80x9d which has some 200 years"" history and has developed as one of the main areas of physiology. Especially in nerves and muscles, the action potential is the most essential event. As it has been revealed that the conduction of excitement is mediated by an electric current, electrophysiology has become one of the most important fields of study for physiologists.
The voltage clamp method was proposed by Cole of the United States in the 1940s. This is a method to keep the membrane potential constant all the time by using at the moment of fluctuation of the membrane potential a feedback circuit which flows an electric current in the direction of suppressing the membrane potential. This voltage clamp method made possible quantitative measurement of the relationship between the membrane potential and ion permeability, and Hodgikin, Huxley and Katz of the United Kingdom used this voltage clamp method to analyze the nature of the membrane conductance of giant axons of Loligo, and made many achievements which would constitute the basis of subsequent studies in neurobiology.
These achievements are reported by Hodgikin, A. L., Huxley, A. F. and Katz in the Journal of Physiology, No.116(1952), pp. 424-44.
This method was further sophisticated by Neher and Sackman subsequently. They succeeded in measuring on a real time basis a current flowing from a live cell to a single-channel molecule. This technique, known as the patch clamp method, is described in detail by Sackman, B. and Neher, E. (eds.), Single-Channel Recording (2nd ed.), Plenum Press, New York (1995).
The central part of a hard glass capillary tube of about 1 mm in diameter is softened by heating with a heater, quickly extended in its lengthwise direction and pulled off to prepare an electrode for recording electric signals. A piece whose pulled-off tip is open and whose diameter is no greater than 1 xcexcm is selected, filled inside with a 3M potassium chloride (KCl) solution by injection, and used as the electrode. By manually penetrating this electrode into each cell, the membrane potential of the cell can be measured.
Most of hormones and other nerve-carried substances convey information to cells via receptors known as seven transmembrane receptors or G protein-coupled receptors. The rapid progress of the Human Genome Project in recent years has resulted in a vast accumulation of information on the base sequences of genes, and it is presumed that there are many so-called xe2x80x9corphanxe2x80x9d receptor genes, whose ligands are unidentified.
The ligands of seven transmembrane receptors are diverse, including hormones, signal transducing substances, cytokine and enzymes, and their molecular variety includes amines, amino acids, peptides, proteins, lipids, nucleic acids and ions. Furthermore, sensory receptors for light, smell and taste are a sort of seven transmembrane receptors, which play an important role in controlling the functions of living organisms. For this reason and because of their deep involvement in diseases, seven transmembrane receptors have been made a major target of powerful medicines. Actually, many of commercially available pharmaceuticals manifest their intended effects when combined with seven transmembrane receptors. Electrophysiometry also provides an important means for screening and determining the ligands of such orphan receptors.
Electrophysiometry is one of the few techniques available for real time measurement of the functions of membrane protein molecules, providing a central approach to receptor proteins. Therefore, electrophysiometry also is an indispensable tool for the development of pharmaceuticals, and its importance is expected to further increase in the future.
The Japanese Published Unexamined Patent Application No. Hei 11-083785 discloses a technique by which Xenopus Oocytes are caused to express histamine receptors, the response of Xenopus Oocytes to histamine is measured, and allergic reactions are detected tissue-specifically.
As stated above, electrophysiometry is indispensable for research on ion channels and the development of pharmaceuticals. However, electrophysiological experiments involve the problem of many troublesome procedures that have to be done manually. First, the worker should prepare glass microelectrodes each with a pulled-off tip whose diameter is no greater than 1 xcexcm by heating and stretching a glass capillary tube. Furthermore, in order to penetrate the glass electrodes into a cell, a micromanipulator should be operated manually.
The micromanipulator, which is an apparatus to hold a glass electrode and manually control minute displacements of the glass electrode, involves the problem of requiring a high level skill to operate. Usually, penetration of a glass electrode into a cell is accomplished by manual operation with a micromanipulator. For this reason, since the glass electrode was devised in the 1940s until even today, electrophysiological measurement has depended heavily on the worker""s craftsmanship.
A breakthrough in the automation of the penetration of Xenopus Oocytes by a glass electrode according to the prior art might be found in the application of image recognition. It is conceivable to determine the position of the membrane surface of a Xenopus Oocyte is determined with a CCD camera from above, and move the glass electrode, driven by a motor or otherwise, to penetrate the oocyte. However, when it is penetrated by the glass electrode, as the membrane surface of the Xenopus Oocyte would be subject to elastic deformation, it would be extremely difficult to check by image recognition from above whether or not the membrane has been accurately penetrated by the glass electrode. Moreover, control of the penetration of the glass electrode by image recognition is an extremely expensive and accordingly unrealistic means of control.
Since current variation responses in an electrophysiological experiment using Xenopus Oocytes or cultured cells may greatly fluctuate from cell to cell, it is necessary to increase the reliability of the data thereby obtained by averaging current responses from many cells. Therefore, in order to obtain fully reliable data, in the electrophysiological experiments each worker should carry out the penetration of a glass electrode into cells many times, resulting in the problem that acquisition of reliable data has to take a long time and a great amount of labor.
An object of the present invention, therefore, is to provide an automatic electrophysiological measuring apparatus and method using Xenopus Oocytes for automatically measuring responses of the cells to the administration of a medicine according to electric signals from a glass electrode penetrating the membranes of the Xenopus Oocytes membrane whose potential is fixed.
The automatic electrophysiological measuring apparatus according to the invention automatically carries out electrophysiological measurement regarding Xenopus Oocytes held in each of a plurality of cells (e.g. 8xc3x9712=96 cells) of a container in which a grounding electrode is arranged or formed by holding and moving the container to position the Xenopus Oocyte in each cell by shifting an XY stage, penetrating the Xenopus Oocyte in each cell with one or two glass electrodes with an inserting means, detecting electric signals emitted from the glass electrode(s) with a detecting means, fixing the membrane potential of the Xenopus Oocytes to a prescribed value with a fixing means, and administering a chemical substance to the Xenopus Oocytes with a microsyringe.
In order to accomplish electrophysiological measurement automatically, the XY stage, inserting means, fixing means and microsyringe are controlled by a control means. The control means detects and distinguishes the contact of the glass electrode(s) with the solution surface in the cell and with the membrane surface of the Xenopus Oocyte and the penetration of the glass electrode(s) into the membrane of the Xenopus Oocyte, controls its (their) penetration into the Xenopus Oocyte and the administration of the chemical substance to the Xenopus Oocyte with the microsyringe, and thereby makes possible automation of the electrophysiological measuring apparatus. Thus, automatic measurement is carried out by measuring the resistance(s) of the glass electrode(s), adjusting the zero-point potential (s) of the glass electrode (s), vibrating the glass electrode(s), controlling the fixation of the membrane potential and administering the chemical substance.
Each cell is shaped in a cone having a semisphere at the tip to hold a Xenopus Oocyte efficiently.
More preferably, the automatic electrophysiological measuring apparatus should be shielded from electromagnetic waves with a shielding means. Also, an optical microscope may be arranged to optically monitor the Xenopus Oocytes held in the cells.
The grounding electrode is molded of an electroconductive metal, with its surface coated with silver chloride, and a grounding wire is individually attached to each cell. The container is molded of agar, with part of which the grounding electrode is brought into contact to make it possible to uniformly ground the plurality of cells. Alternatively, the grounding electrode may as well be moved on a single axis in the solution held in the cell to ground each cell independently of others.
More specifically Xenopus Oocyte holding plates, in each of whose cells 8xc3x9712=96 Xenopus Oocytes are regularly arranged, are mounted on the XY stage. To each cell in which one Xenopus Oocyte is held, a grounding electrode is independently attached. One Xenopus Oocyte positioned by the XY stage at the center of the field of vision of a stereoscopic microscope is automatically penetrated by two glass electrodes by a motor-driven movement. The XY stage, the motor to drive the glass electrodes, the motor to drive the microsyringe for administering the ligand and the electrophysiological measuring apparatus are collectively controlled from outside by a control computer on the basis of electric signals from the glass electrodes, and automatic electrophysiological measurement regarding the plurality of Xenopus Oocytes is thereby enabled. The measured responses of the Xenopus Oocytes to the current are recorded by a measurement recording computer.
By the automatic electrophysiological measuring method according to the invention, electrophysiological measurement regarding Xenopus Oocytes is automatically carried out by holding on an XY stage a container having a plurality of cells each holding a Xenopus Oocyte, in each of the cells a grounding electrode being arranged or formed, moving the XY stage to position the Xenopus Oocyte in a prescribed cell, penetrating one or two glass electrodes into the Xenopus Oocyte in the prescribed cell, detecting electric signals emitted from the glass electrode(s), fixing the membrane potential of the Xenopus Oocyte to a prescribed value, and administering a chemical substance to the Xenopus Oocyte with a microsyringe. In order to accomplish electrophysiological measurement automatically, the movement of the XY stage, the penetration of the glass electrode(s) into the Xenopus Oocyte, the fixation of the membrane potential of the Xenopus Oocyte and the administration of the chemical substance to the Xenopus Oocyte are controlled on the basis of electric signals emitted from the glass electrode(s). These controls are accomplished by detecting and distinguishing the contact of the glass electrode(s) with the solution surface in the cell and with the membrane surface of the Xenopus Oocyte and the penetration of the glass electrode(s) into the membrane of the Xenopus Oocyte, thereby making possible automation of the electrophysiological measuring apparatus.
According to the invention, there is provided an automatic electrophysiological measuring apparatus which automatically penetrates glass electrodes into Xenopus Oocytes membranes, holds the membrane potentials and automatically measures reactions to the administration of a medicine. The principle of the invention will be explained below with reference to FIG. 3. First, a glass electrode 103 in the air is moved toward a Xenopus Oocyte 601 held in a cell. When the glass electrode 103 comes into contact with a solution surface 603 to become electrically connected to a grounding electrode 604, the potential of the glass electrode points to around 0 mV. When the glass electrode 103 is further moved to come into contact with the membrane surface of the Xenopus Oocyte 601, the potential of the glass electrode 103 points to around xe2x88x925 mV. Further, as the other glass electrode 102 is moved to penetrate the membrane, its potential points to around xe2x88x9220 mV. These changes in the potentials of the glass electrodes are picked up by the control computer to identify the relative positions of the glass electrode 102 and the Xenopus Oocyte 601 to automate the sequence of electrophysiological measuring to penetrate the glass electrode 102 into the Xenopus Oocyte 601, hold its membrane potential and administer the medicine.
The automatic electrophysiological measuring apparatus according to the invention using Xenopus Oocytes can enhance the reliability of acquired data by automating the electrophysiological measurement and increasing the frequency of measurement while reducing the workload on the measuring personnel. Automation also enables the measurement of many samples to be accomplished at high speed and the processing of many tasks of electrophysiological measurement in a short period of time. Furthermore, it enables the unknown function of a gene whose base sequence is already known to be screened.